Simultaneous analysis of a large number of biological samples is useful in flow cytometry, DNA sequencing, liquid chromatography, oligonucleotide analysis, zone electrophoresis of proteins, as well as other electrophoretic techniques. In particular, rapid DNA analysis is of importance in the Human Genome Project, which is an attempt to identify the sequence of bases (dideoxynucleotides) in human DNA.
One technique that has been applied to the sequencing of DNA is capillary electrophoresis. In capillary electrophoresis, an appropriate solution is polymerized or gelled to form a porous matrix in a fused silica capillary tube of internal dimensions in the order of 50 .mu.m. An electric filed is applied across the matrix. Fragments of sample DNA injected into one end of the capillary tube migrate through the matrix under the effect of the electric field at speeds that depend on the length of the fragment. Hence, different length fragments arrive at a detection part of the capillary at different times. The dideoxynucleotide at one end of the fragment may be labelled with a fluorescent marker during a reaction step. The fluorescent marker is associated with the terminating dideoxynucleotide. When the fragment passes through a beam of light from a laser in the detection zone, the fluorescent marker fluoresces and the fluorescence may be detected as an electric signal. The intensity of the electric signal depends on the amount of fluorescent marker present in the matrix in the detection zone. The dideoxynucleotide at the end of the fragment may then be identified by a variety of methods. As different length fragments migrate through the matrix under the applied field, a profile of the fragments may be obtained.
The use of three different DNA sequencing techniques is set out in Swerdlow, H. et al, Three DNA sequencing. Methods Using Capillary Gel Electrophoresis and Laser Induced Fluorescence, Anal. Chem., 53, 2835-2841, Dec. 15, 1991, and the references cited therein. In the Tabor and Richardson technique (one spectral channel sequencing), a single fluorescent marker is used, and the amount of dideoxynucleotide in the reaction mixture is varied so that each base of the DNA fragment may be identified with a particular fluorescent peak height. For example, the concentration of dideoxynucleotides might be varied in the ratio of 8:4:2:1. The variation, in fluorescence intensity with time will then identify the sequence of bases. In the DuPont system (two spectral channel sequencing), succinylfluorescein dyes are used to label four dideoxynucleotides. A single wavelength (488 nm) is used to excite fluorescence from the dyes. Emission is distributed between two spectral channels centered at 510 and 540 nm. The ratio of the fluorescent intensity in the two spectral channels is used to identify the terminating dideoxynucleotide. In the Applied Biosystems system (four spectral channel sequencing), four dyes (FAM, JOE, TAMRA and ROX) are used to label primers to be used with each dideoxynucleotide reaction. Two lines from an argon laser (514.5 and 488 nm) are used to excite fluorescence. Interference filters are used to isolate emission at 540, 560, 580 and 610 nm and peaks of the resulting four electrical signal profiles are used to identify the bases.
Application of capillary electrophoresis to DNA analysis is complicated by the scattering of light from the porous matrix and the capillary walls. For this reason, there has been proposed use of a sheath flow cuvette in which a sample stream of DNA is injected under laminar flow conditions in the center of a surrounding sheath stream, generally of the same refractive index. Such a cuvette is described in Swerdlow H., et al, Capillary Gel Electrophoresis for DNA Sequencing: Laser Induced fluorescence detection with the sheath flow cuvette, Journal of Chromatography, 516, 1990, 61-67.
However, the above described methods of DNA sequencing using capillary electrophoresis have used single capillaries and rapid DNA sequencing and other biological process requiring simultaneous analysis of sample streams require use of multiple capillary systems.
One such multiple capillary system is described in Huang et al, Capillary Array Electrophoresis Using Laser Excited Confocal Fluorescence Detection, Anal. Chem. 64, 967-972, Apr. 15, 1992. In the Huang device, multiple capillaries lying side by side in a flat array holder are sequentially scanned by a laser beam and fluorescence detected from the capillaries using a photomultiplier tube. Such a device suffers from the same difficulties as with a single capillary that is scanned with a laser, namely that there is light scatter from the capillary walls and interfaces between the matrix and capillary.
The inventors have therefore proposed a multiple capillary analyzer that allows detection of light from multiple capillaries with a reduced number of interfaces through which light must pass in detecting light emitted from a sample being analyzed.
In one aspect of the invention, there is provided a multiple capillary analyzer using a modified sheath flow cuvette. A linear array of capillaries is introduced into a rectangular flow chamber. Sheath fluid draws individual sample streams through the cuvette. The capillaries are closely and evenly spaced and held by a transparent retainer in a fixed position in relation to an optical detection system. Collimated sample excitation radiation is applied simultaneously across the ends of the capillaries in the retainer. Light emitted from the excited sample is detected by the optical detection system.
In a further aspect of the invention, the retainer is provided by a transparent chamber having inward slanting end walls. The capillaries are wedged into the chamber. One sideways dimension of the chamber is equal to the diameter of the capillaries and one end to end dimension varies from, at the top of the chamber, slightly greater than the sum of the diameters of the capillaries to, at the bottom of the chamber, slightly smaller than the sum of the diameters of the capillaries.
In a still further aspect of the invention, the optical system utilizes optic fibres to deliver light to individual photodetectors, one for each capillary tube. A filter or wavelength division demultiplexer may be used for isolating fluorescence at particular bands.
In a still further aspect of the invention, the array may be rectangular, including square, or the like formed of plural rows of capillaries terminating at different levels in a rectangular sheath flow cuvette. A rectangular array of lenses receives light emitted, scattered or reflected from samples emerging from the capillaries and the light is then converted to electrical signals and processed.